High-purity large-scale preparation of stannsoporfin

ABSTRACT

Large scale (bulk) compositions comprising high-purity stannsoporfin are disclosed, as well as methods of synthesizing such compositions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 14/793,921, filed Jul. 8, 2015, which is a continuation of U.S.application Ser. No. 14/460,080, filed Aug. 14, 2014, now U.S. Pat. No.9,107,927, issued Aug. 18, 2015, which is a continuation of U.S.application Ser. No. 14/019,513, filed Sep. 5, 2013, now U.S. Pat. No.8,835,416, issued Sep. 16, 2014, which is a continuation of U.S.application Ser. No. 12/968,651, filed Dec. 15, 2010, now U.S. Pat. No.8,530,458, issued Sep. 10, 2013, which is a continuation of U.S.application Ser. No. 11/867,559, filed Oct. 4, 2007, now U.S. Pat. No.7,960,371, issued Jun. 14, 2011, which claims the benefit of U.S.Provisional Appln. No. 60/849,641, filed Oct. 4, 2006 and U.S.Provisional Appln. No. 60/904,601, filed Feb. 28, 2007.

TECHNICAL FIELD

This invention pertains to methods for synthesizing stannsoporfin (tin(IV) mesoporphyrin IX dichloride) in large quantities at high purity,and the compositions so produced.

BACKGROUND

Stannsoporfin, or tin (IV) mesoporphyrin IX dichloride, is an inhibitorof the enzyme heme oxygenase. Stannsoporfin has been proposed fortherapeutic use in several diseases, such as infant hyperbilirubinemia(U.S. Pat. Nos. 4,657,902; 4,668,670; WO 94/28906) and psoriasis (U.S.Pat. No. 4,782,049). Because of this pharmaceutical utility, methods ofpreparing stannsoporfin are of great interest.

Infant hyperbilirubinemia (also known as infant jaundice or neonatalhyperbilirubinemia occurs in a newborn when the liver is unable toconjugate bilirubin so it can be excreted at a rate commensurate withbilirubin formation. Bilirubin comes from the release of heme as part ofthe physiological conversion from fetal to adult hemoglobin at birth.The enzyme heme oxygenase oxidizes heme to biliverdin; the enzymebiliverdin reductase then reduces the biliverdin to bilirubin. Bilirubinat high serum levels is a neurotoxic substance. In adult humans, theliver rapidly converts bilirubin into a conjugated, excretable form. Innewborn humans, however, the the liver is still developing, and uptakeand conjugation by the liver is not as efficient as in adults.Additionally, hemolysis may be taking place at a greater relative ratethan in adults. All of these factors can lead to excessive bilirubin inthe infant. For some infants, high serum levels of bilirubin can havedetrimental physiological consequences. Bilirubin is yellow, and infantswith excess bilirubin appear jaundiced, having a yellow tinge to theirskin and to the whites of their eyes.

Infants who have highly elevated serum levels of bilirubin are at riskof developing kernicterus, a rare but potentially devastatingneurological disorder which can result in severe life-long disabilitiesand complications such as athetosis, hearing loss, vision problems, anddental problems. (See Centers for Disease Control and PreventionWorld-Wide-Web.cdc.gov/ncbddd/dd/kernicterus.htm.) Accordingly, infantsshould be carefully monitored after birth, and therapeutic interventionshould be commenced if an infant's bilirubin level is excessive. TheAmerican Academy of Pediatrics has published a Clinical PracticeGuideline for evaluating newborns for hyperbilirubinemia and treatingat-risk newborns; see Pediatrics 114:297-316 (2004). As health-carecosts have risen in the United States, seemingly healthy newborns andtheir mothers are discharged rapidly, sometimes as quickly as 24 to 48hours after birth. However, it is believed that this practice may havecontributed to an increase in cases of kernicterus, which had beenvirtually eliminated from developed countries; see Hansen TWR, ActaPaediatr. 89:1155-1157 (2000)). Because early discharge can delay thedetection of jaundice and hyperbilirubinemia in infants, effective meansof treating hyperbilirubinemia rapidly are desirable. The unique medicalstatus of the newborn also requires that any means of treatment be assafe as possible, as side effects that are tolerable in adults may becompletely unacceptable in neonates.

Currently approved and commonly used treatments for hyperbilirubinemiainclude phototherapy and exchange transfusion. Phototherapy involvesirradiating the newborn with light in the 430 to 490 nm range (bluelight). The light converts bilirubin into lumirubin and photobilirubin,which are more readily excreted by the infant, and thus can result in areduction of bilirubin levels.

Stannsoporfin (tin (IV) mesoporphyrin IX dichloride) has beendemonstrated to be of therapeutic value in treating hyperbilirubinemia;see Valaes et al., Pediatrics 93:1-11 (1994) and Kappas et al.,Pediatrics, 95:468-474 (1995). Other indications in which stannsoporfincan be used are disclosed in U.S. Pat. No. 4,692,440 (to increase therate of heme excretion), WO 89/02269 (to counteract the toxicity ofcancer therapy), U.S. Pat. No. 4,782,049 (to treat psoriasis) and otherpublications.

U.S. Pat. No. 6,818,763, U.S. Patent Application Publication2004/0210048, and U.S. patent application Ser. No. 11/096,359 disclosemethods of synthesizing stannsoporfin. However, it is still desirable todevelop methods to produce stannsoporfin at higher purity, due to thetherapeutic advantages of using as pure a substance as possible and alsodue to the stringent requirements of regulatory agencies.

The current application discloses methods of synthesizing stannsoporfinat a heretofore unachieved level of purity, as well as large-scalepreparations of pure stannsoporfin. The current application alsodiscloses a new method of insertion of tin and other metals intoporphyrin rings. This new method can significantly decrease the timerequired for synthesis of stannsoporfin.

DISCLOSURE OF THE INVENTION

The current invention embraces, in certain aspects, high-puritystannsoporfin in large scale (bulk) quantity, and methods for makingsuch compositions of stannsoporfin. The invention also embraces othersynthetic methods and chemical compositions as disclosed herein.

In one embodiment, the invention embraces a composition of mattercomprising high-purity stannsoporfin in large scale (or bulk) quantity.In another embodiment, the invention embraces a composition of mattercomprising high-purity stannsoporfin in large scale (or bulk) quantitywhen produced in a single batch, that is, a single-batch large scale (orbulk) high-purity amount of stannsoporfin. The high-purity stannsoporfincan be at least about 97% pure, at least about 98% pure, at least about98.5% pure, at least about 99% pure, at least about 99.5% pure, or atleast about 99.8% pure. The amount of any single impurity in thehigh-purity stannsoporfin can be less than about 0.1%, less than about0.09%, less than about 0.08%, or about 0.7% or less; in anotherembodiment, the any single impurity is any single product-relatedimpurity. The large-scale (bulk) amount of stannsoporfin can be at leastabout 10 grams, at least about 25 grams, at least about 50 grams, atleast about 100 grams, at least about 200 grams, at least about 500grams, at least about 1.0 kg, at least about 2.0 kg, or at least about5.0 kg. In one embodiment, the high-purity stannsoporfin as variouslydescribed above is produced in a single batch.

In an alternative embodiment, the large scale quantity of stannsoporfinis at least about 97% pure, at least about 98% pure, at least about98.5% pure, at least about 99% pure, at least about 99.5% pure, or atleast about 99.8% pure, and has no impurity present in an amount greaterthan about 0.2%, and more preferably has no impurity present in anamount greater than 0.15%, and still more preferably has no impuritypresent in an amount greater than 0.12%. In one embodiment, thehigh-purity stannsoporfin is produced in a single batch.

In additional embodiments, the amount of palladium impurities present inthe large-scale amount of high-purity stannsoporfin is less than about20 ppm, less than about 15 ppm, less than about 10 ppm, or less thanabout 5 ppm. In one embodiment, the high-purity stannsoporfin isproduced in a single batch.

In another embodiment, the invention embraces a method of makinghigh-purity stannsoporfin on a large scale, comprising the steps of: a)exposing a metallic hydrogenation catalyst to a hydrogen atmosphere toform pre-hydrogenated catalyst; and b) contacting hemin with thepre-hydrogenated catalyst and maintaining the hemin and catalyst underone or more combinations of temperature, hydrogen pressure, and timesufficient to remove iron from the hemin and reduce the vinyl groups ofthe hemin to ethyl groups, thus forming mesoporphyrin IX. In anotherembodiment, the invention embraces a method of making high-puritystannsoporfin on a large scale, comprising the steps of a) exposing ametallic hydrogenation catalyst to a hydrogen atmosphere to formpre-hydrogenated catalyst; b) contacting hemin with the pre-hydrogenatedcatalyst and maintaining the hemin and catalyst under one or morecombinations of temperature, hydrogen pressure, and time sufficient toremove iron from the hemin and reduce the vinyl groups of the hemin toethyl groups, thus forming mesoporphyrin IX; and c) reactingmesoporphyrin IX with a tin (II) salt to form stannsoporfin using acontrolled rate of oxidation. In one embodiment, the metallichydrogenation catalyst comprises palladium, palladium on carbon,platinum, platinum on carbon, nickel, or nickel-aluminum catalyst. Inanother embodiment, the metallic hydrogenation catalyst is palladium. Inanother embodiment, the metallic hydrogenation catalyst is palladium oncarbon. The method can produce a large-scale amount of high-puritystannsoporfin in a single batch.

In another embodiment, the invention embraces a method of makinghigh-purity stannsoporfin on a large scale, comprising the steps of: a)exposing a metallic hydrogenation catalyst to a hydrogen atmosphere toform pre-hydrogenated catalyst; b) contacting hemin with thepre-hydrogenated catalyst and maintaining the hemin and catalyst underone or more combinations of temperature, hydrogen pressure, and timesufficient to remove iron from the hemin and reduce the vinyl groups ofthe hemin to ethyl groups, thus forming mesoporphyrin IX; and c)reacting mesoporphyrin IX with tin (II) oxide to form stannsoporfin. Inone embodiment, the metallic hydrogenation catalyst comprises palladium,palladium on carbon, platinum, platinum on carbon, nickel, ornickel-aluminum catalyst. In another embodiment, the metallichydrogenation catalyst is palladium. In another embodiment, the metallichydrogenation catalyst is palladium on carbon. The method can produce alarge-scale amount of high-purity stannsoporfin in a single batch.

In another embodiment, the invention embraces a method of makingmesoporphyrin IX, comprising the steps of: a) exposing a palladium oncarbon catalyst to a hydrogen atmosphere to form pre-hydrogenatedpalladium catalyst; and b) contacting hemin with the pre-hydrogenatedcatalyst and maintaining the hemin and catalyst under one or morecombinations of temperature, hydrogen pressure, and time sufficient toremove iron from the hemin and reduce the vinyl groups of the hemin toethyl groups, thus forming mesoporphyrin IX. In additional embodiments,step b) is carried out at about 80 to 100° C., preferably at about 85 to90° C., with hydrogen pressure at about 50 to 70 psi, preferably atabout 55 to 60 psi, for about 1 to 3 hours, preferably about 1 to 1.5hours; then at about 40 to 60° C. , preferably about 45 to 50° C., withhydrogen pressure at about 50 to 70 psi, preferably at about 55 to 60psi, for about 18 to 48 hours, preferably about 24 hours.

In another embodiment, the invention embraces a preparation of readilyfilterable mesoporphyrin IX dihydrochloride, wherein at least about 10grams can be filtered in less than about 90 minutes, less than about 60minutes, less than about 45 minutes, less than about 35 minutes, lessthan about 25 minutes, or less than about 10 minutes, from a solutionwhere the amount of solvent is present in at least about a 50-to-1 ratioby weight to the amount of mesoporphyrin IX dihydrochloride. In anotherembodiment, the invention embraces a preparation of readily filterablemesoporphyrin IX dihydrochloride, wherein at least about 1000 grams canbe filtered in less than about 1 day, less than about 12 hours, lessthan about 6 hours, less than about 4 hours, less than about 3 hours, orless than about 2 hours, from a solution where the amount of solvent ispresent in at least about a 50-to-1 ratio by weight to the amount ofmesoporphyrin IX dihydrochloride. In one embodiment, the solvent is amixture of water, hydrochloric acid, and formic acid; the hydrochloricacid can be about 31% hydrochloric acid prior to mixing.

In another embodiment, the invention embraces a method of making areadily filterable mesoporphyrin IX dihydrochloride preparation,comprising the step of adding an aqueous solution of hydrochloric acidto a solution of mesoprophyrin IX formate in formic acid. In oneembodiment, the concentration of hydrochloric acid in the aqueoussolution is about 0.5 to 2.0N. In another embodiment, the concentrationof hydrochloric acid in the aqueous solution is about 0.75 to 1.25N. Inanother embodiment, the concentration of hydrochloric acid in theaqueous solution is about 1.0N.

In another embodiment the invention embraces a method of inserting tininto mesoporphyrin IX, comprising reacting the mesoporphyrin IX with atin salt in the absence of a proton scavenger.

In another embodiment, the invention embraces a method of inserting tininto mesoporphyrin IX, comprising reacting the mesoporphyrin IX with atin salt at a controlled rate of oxidation. In one embodiment, themesoporphyrin IX is reacted with a tin salt in a reaction vessel havinga headspace, and the rate of oxidation is controlled by introducing anoxygen-containing gas into the headspace of the reaction vessel. Inanother embodiment, the oxygen-containing gas introduced into theheadspace of the reaction vessel is about 3 to 22% oxygen in an inertgas, such as nitrogen. In another embodiment, the oxygen-containing gasintroduced into the headspace of the reaction vessel is air. In anotherembodiment, the oxygen-containing gas introduced into the headspace ofthe reaction vessel is about 4 to 15% oxygen in an inert gas, such asnitrogen. In another embodiment, the oxygen-containing gas introducedinto the headspace of the reaction vessel is about 5 to 10% oxygen in aninert gas, such as nitrogen. In another embodiment, theoxygen-containing gas introduced into the headspace of the reactionvessel is about 6% oxygen in an inert gas, such as nitrogen. In anotherembodiment, the oxygen-containing gas introduced into the headspace ofthe reaction vessel is about 5% oxygen in nitrogen.

In any of the above embodiments, the large scale amount of high-puritystannsoporfin can be produced in a single batch.

In any of the above embodiments, the reactants, intermediates, and/orproducts can undergo additional steps of purification. In someembodiments, the additional purification comprises treating thereactant, intermediate, or product with diatomaceous earth and/oractivated carbon. In one embodiment, the treating of the reactant,intermediate, or product with diatomaceous earth and/or activated carboncomprises dissolving or suspending the reactant, intermediate, and/orproduct in a solvent, adding diatomaceous earth and/or activated carbon,filtering off the diatomaceous earth and/or activated carbon, andrecovering the reactant, intermediate, or product from the filtrate. Insome embodiments, the additional purification comprises triturating thereactant, intermediate, or product with hot acid, such as about 0.1 to6N HCl in water, preferably about 3N HCl in water. In some embodiments,one, two, or three of the steps of treating with diatomaceous earth,treating with activated carbon, and triturating with hot acid areperformed sequentially, in any order, and can be repeated as desired.

In another embodiment, the invention embraces a method of inserting ametal into a porphyrin compound or a salt thereof using a metal oxide,in another embodiment, the metal cation of the metal oxide is in anintermediate oxidation state. In another embodiment, the porphyrincompound is a mesoporphyrin IX or salt thereof, or a protoporphyrin orsalt thereof, or a hematoporphyrin or salt thereof, or adeuteroporphyrin or salt thereof. In another embodiment, the porphyrincompound is mesoporphyrin IX or a salt thereof. In another embodiment,the porphyrin compound is mesoporphyrin IX dihydrochloride. In anotherembodiment, the resulting product is a metallated porphyrin or a saltthereof. In another embodiment, the resulting product is a metallatedmesoporphyrin or a salt thereof or a metallated protoporphyrin or a saltthereof or a metallated hematoporphyrin or a salt thereof or ametallated deuteroporphyrin or a salt thereof. In other embodiments, themetal oxide is selected from tin oxide, zinc oxide, copper oxide,cadmium oxide, cobalt oxide, chromium oxide, iron oxide, aluminum oxide,titanium oxide, nickel oxide, manganese oxide, silver oxide, gold oxide,vanadium oxide, platinum oxide, antimony oxide, or arsenic oxide. Inother embodiments, the metal oxide is selected from tin (II) oxide, zinc(II) oxide, copper (I) oxide, copper (II) oxide, cadmium (II) oxide,cobalt (II) oxide, cobalt (III) oxide, cobalt (IV) oxide, Co₃O₄,chromium (II) oxide, chromium (III) oxide, chromium (IV) oxide, chromium(V) oxide, chromium (VI) oxide, iron (II) oxide, iron (III) oxide,Fe₃O₄, aluminum (III) oxide, titanium (II) oxide, titanium (III) oxide,titanium (IV) oxide, nickel (II) oxide, manganese (II) oxide, manganese(III) oxide, manganese (IV) oxide, manganese (VII) oxide, silver (I)oxide, silver (II) oxide, gold (I) oxide, gold (III) oxide, vanadium(II) oxide, vanadium (III) oxide, vanadium (IV) oxide, vanadium (V)oxide, platinum (II) oxide, platinum (IV) oxide, antimony (III) oxide,antimony (IV) oxide, antimony (V) oxide, arsenic (III) oxide, or arsenic(V) oxide. In other embodiments, the resulting product is a tinporphyrin, zinc porphyrin, copper porphyrin, cadmium porphyrin, cobaltporphyrin, chromium porphyrin, iron porphyrin, aluminum porphyrin,titanium porphyrin, nickel porphyrin, manganese porphyrin, silverporphyrin, gold porphyrin, vanadium porphyrin, platinum porphyrin,antimony porphyrin, arsenic porphyrin, or a salt thereof. In otherembodiments, the resulting product is a tin mesoporphyrin, zincmesoporphyrin, copper mesoporphyrin, cadmium mesoporphyrin, cobaltmesoporphyrin, chromium mesoporphyrin, iron mesoporphyrin, aluminummesoporphyrin, titanium mesoporphyrin, nickel mesoporphyrin, manganesemesoprophyrin, silver mesoporphyrin, gold mesoporphyrin, vanadiummesoporphyrin, platinum mesoporphyrin, antimony mesoporphyrin, arsenicmesoporphyrin, or a salt thereof. In other embodiments, the resultingproduct is a tin mesoporphyrin IX, zinc mesoporphyrin IX, coppermesoporphyrin IX, cadmium mesoporphyrin IX, cobalt mesoporphyrin IX,chromium mesoporphyrin IX, iron mesoporphyrin IX, aluminum mesoporphyrinIX, titanium mesoporphyrin IX, nickel mesoporphyrin IX, manganesemesoporphyrin IX, silver mesoporphyrin IX, gold mesoporphyrin IX,vanadium mesoporphyrin IX, platinum mesoporphyrin IX, antimonymesoporphyrin IX, arsenic mesoporphyrin IX, or a salt thereof. In otherembodiments, the resulting product is a tin protoporphyrin, zincprotoporphyrin, copper protoporphyrin, cadmium protoporphyrin, cobaltprotoporphyrin, chromium protoporphyrin, iron protoporphyrin, aluminumprotoporphyrin, titanium protoporphyrin, nickel protoporphyrin,manganese protoporphyrin, silver protoporphyrin, gold protoporphyrin,vanadium protoporphyrin, platinum protoporphyrin, antimonyprotoporphyrin, arsenic protoporphyrin, or a salt thereof. In otherembodiments, the resulting product is a tin hematoporphyrin, zinchematoporphyrin, copper hematoporphyrin, cadmium hematoporphyrin, cobalthematoporphyrin, chromium hematoporphyrin, iron hematoporphyrin,aluminum hematoporphyrin, titanium hematoporphyrin, nickelhematoporphyrin, manganese hematoporphyrin, silver hematoporphyrin, goldhematoporphyrin, vanadium hematoporphyrin, platinum hematoporphyrin,antimony hematoporphyrin, arsenic hematoporphyrin, or a salt thereof. Inother embodiments, the resulting product is a tin deuteroporphyrin, zincdeuteroporphyrin, copper deuteroporphyrin, cadmium deuteroporphyrin,cobalt deuteroporphyrin, chromium deuteroporphyrin, irondeuteroporphyrin, aluminum deuteroporphyrin, titanium deuteroporphyrin,nickel deuteroporphyrin, manganese deuteroporphyrin, silverdeuteroporphyrin, gold deuteroporphyrin, vanadium deuteroporphyrin,platinum deuteroporphyrin, antimony deuteroporphyrin, arsenicdeuteroporphyrin, or a salt thereof.

In another embodiment, the invention embraces a method of inserting tininto a porphyrin compound or a salt thereof using tin (II) oxide. Inanother embodiment, the porphyrin compound is a mesoporphyrin or saltthereof, or a protoporphyrin or salt thereof, or a hematoporphyrin orsalt thereof. In another embodiment, the porphyrin compound ismesoporphyrin IX or a salt thereof. In another embodiment, the porphyrincompound is mesoporphyrin IX dihydrochloride. In another embodiment, theresulting product is a tin (IV) porphyrin or a salt thereof. In anotherembodiment, the resulting product is a tin (IV) mesoporphyrin or a saltthereof or tin (IV) protoporphyrin or a salt thereof or tin (IV)hematoporphyrin or a salt thereof. In another embodiment, the resultingproduct is tin (IV) mesoporphyrin IX or a salt thereof.

In another embodiment, the invention embraces a method of inserting tininto a porphyrin compound or a salt thereof by providing a porphyrincompound or salt thereof, providing tin (II) oxide, and contacting thetin (II) oxide with the porphyrin compound or salt thereof under acidicconditions, whereby the tin (II) oxide inserts into the porphyrin ringto yield a tin (IV) porphyrin compound. In another embodiment, the tin(II) oxide is dissolved or suspended in acetic acid or formic acid,preferably acetic acid. In another embodiment, the porphyrin compound orsalt thereof is dissolved or suspended in formic acid or acetic acid,preferably formic acid. In another embodiment, the equivalent ratio ofthe total amount of tin (II) oxide used to the total amount of porphyrincompound or salt thereof used is about two to six, preferably aboutfour. In another embodiment, the solution or suspension of the porphyrincompound or salt thereof is added dropwise to the tin (II) oxidesolution. The dropwise addition can take place over a period of aboutthree to nine hours, preferably over about six hours. The tin II oxidesolution or suspension is maintained at a temperature of about 25-115°C., preferably about 50-75° C., more preferably about 60-65° C.

In another embodiment, the invention embraces a method of inserting tininto a porphyrin compound or a salt thereof (such as mesoporphyrin IX ora salt thereof, such as mesoporphyrin IX dihydrochloride) by providing amesoporphyrin compound or salt thereof (such as mesoporphyrin IX or asalt thereof, such as mesoporphyrin IX dihydrochloride), providing tin(II) oxide, and contacting the tin (II) oxide with the mesoporphyrincompound or salt thereof (such as mesoporphyrin IX or a salt thereof,such as mesoporphyrin IX dihydrochloride) under acidic conditions,whereby the tin (II) oxide inserts into the porphyrin ring (such as amesoporphyrin IX ring) to yield a tin (IV) porphyrin compound. Inanother embodiment, the tin (II) oxide is dissolved or suspended inacetic acid or formic acid, preferably acetic acid. In anotherembodiment, the mesoporphyrin compound or salt thereof (such asmesoporphyrin IX or a salt thereof, such as mesoporphyrin IXdihydrochloride) is dissolved or suspended in formic acid or aceticacid, preferably formic acid. In another embodiment, the equivalentratio of the total amount of tin (II) oxide used to the total amount ofmesoporphyrin compound or salt thereof (such as mesoporphyrin IX or asalt thereof, such as mesoporphyrin IX dihydrochloride) used is abouttwo to six, preferably about four. In another embodiment, the solutionor suspension of the porphyrin compound or salt thereof (suchmesoporphyrin IX or a salt thereof, such as mesoporphyrin IXdihydrochloride) added dropwise to the tin (II) oxide solution. Thedropwise addition can take place over a period of about three to ninehours, preferably over about six hours. The tin (II) oxide solution orsuspension is maintained at a temperature of about 25-115° C.,preferably about 50-75° C., more preferably about 60-65° C. Aftercompletion of the dropwise addition, the reaction mixture can bemaintained at a temperature of about 60 to 65° C. for about 18 to 24additional hours. The reaction mixture can be cooled and filtered afterthe additional reaction time.

In another embodiment, the invention embraces a method for producing atin (IV) porphyrin compound or salt thereof comprising a) preparing asolution or suspension of an unmetallated porphyrin compound or a saltthereof; b) preparing a solution or suspension of tin (II) oxide,wherein steps (a) and (b) can occur in any order or simultaneously; andc) contacting the solution or suspension of tin (II) oxide with thesolution or suspension of unmetallated porphyrin compound or saltthereof under conditions suitable to form the tin (IV) porphyrincompound or salt thereof. The solution or suspension of tin (II) oxideand the solution or suspension of unmetallated porphyrin compound orsalt thereof can be independently prepared with formic acid or aceticacid; for example, the solution or suspension of tin (II) oxide can beprepared with acetic acid, and the solution or suspension ofunmetallated porphyrin compound or salt thereof can be prepared withformic acid. The unmetallated porphyrin compound is selected frommesoporphyrins, protoporphyrins, hematoporphyrins, and salts thereof,such as mesoporphyrin IX or a salt thereof, such as mesoporphyrin IXdihydrochloride. The contacting step c) can comprise adding the solutionor suspension of unmetallated porphyrin compound or salt thereof in adropwise manner to the solution or suspension of tin (II) oxide underconditions suitable to form the tin (IV) porphyrin compound or saltthereof. The adding in a dropwise manner can completed within about 3 to9 hours, such as about 6 hours. The solution or suspension of tin (II)oxide can be maintained at a temperature of about 60 to 65° C. duringthe adding in a dropwise manner. After completion of the adding of thesolution or suspension of unmetallated porphyrin compound or saltthereof in a dropwise manner to the solution or suspension of tin (II)oxide, the reaction mixture can be maintained at a temperature of about60 to 65° C. for about 18 to 24 additional hours. The reaction mixturecan be cooled and filtered after the additional reaction time. Inanother embodiment, the method for producing a tin (IV) porphyrincompound or salt thereof is performed in the absence of a protonscavenger or proton sponge.

The tin (IV) mesoporphyrin produced by any of the methods describedabove can undergo additional steps of purification. In some embodiments,the additional purification comprises treating the tin (IV)mesoporphyrin with diatomaceous earth and/or activated carbon. In oneembodiment, the treating of the tin (IV) mesoporphyrin with diatomaceousearth and/or activated carbon comprises dissolving or suspending the tin(IV) mesoporphyrin in a solvent, adding diatomaceous earth and/oractivated carbon, filtering off the diatomaceous earth and/or activatedcarbon, and recovering the tin (IV) mesoporphyrin from the filtrate. Insome embodiments, the additional purification comprises triturating thetin (IV) mesoporphyrin with hot acid, such as about 0.1 to 6N HCl inwater, preferably about 3N HCl in water, at a temperature of about 60 to95° C., preferably about 80 to 95° C., more preferably about 85 to 90°C. In some embodiments, one, two, or all three of the steps of treatingwith diatomaceous earth, treating with activated carbon, and trituratingwith hot acid art performed sequentially, in any order, and can berepeated as desired.

In another embodiment, the invention embraces stannsoporfin as producedby any of the processes described herein.

In another embodiment, the invention embraces high-purity stannsoporfinin large scale (or bulk) quantity, wherein said high-puritystannsoporfin is stable for at least about three months or at leastabout six months under storage. In another embodiment, the high-puritystannsoporfin in large quantity is prepared as a single batch. Inanother embodiment, the invention embraces high-purity stannsoporfin inlarge scale (or bulk) quantity, wherein said high-purity stannsoporfinretains its high purity for at least about three months or at leastabout six months under storage. In another embodiment, the storageconditions are about 25° C. and about 60% relative humidity. In anotherembodiment, the storage conditions are about 40° C. and about 75%relative humidity. In another embodiment, the stannsoporfin is stored ina polyethylene bag. In another embodiment, the stannsoporfin is storedin a polyethylene bag within another polyethylene bag. In anotherembodiment, the double-bagged stannsoporfin is stored in a high-densitypolyethylene drum. In another embodiment, each polyethylene bag is about4 mils thick (about 4/1000 of an inch, or about 0.1 millimeter).

In another embodiment, the invention embraces a method of treatinginfant hyperbilirubinemia, comprising administering stannsoporfin to apatient in need of said treatment, where the stannsoporfin was producedin high purity and on a large scale. In another embodiment, thestannsoporfin is produced as a single batch.

In another embodiment, the invention embraces a method of preventinginfant hyperbilirubinemia, comprising administering stannsoporfin to apatient in need of said prevention, where the stannsoporfin was producedin high purity and on a large scale. In another embodiment, thestannsoporfin is produced as a single batch.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment of the current invention, stannsoporfin is prepared inlarge quantity at high purity. In another embodiment of the currentinvention, stannsoporfin is prepared in large quantity at high purity,wherein the large quantity is prepared as a single batch. Stannsoporfin(tin (IV) mesoporphyrin IX dichloride; Chemical Abstracts RegistryNumber 106344-20-1) is also known by the trade name Stanate®, which is aregistered trademark of InfaCare Pharmaceutical Corp., Plymouth Meeting,Pa. Stannsoporfin has the following structure:

having molecular formula C₃₄H₃₆Cl₂N₄O₄Sn and molecular weight 754.29.

By “large quantity,” “large scale,” or “bulk” is meant at least about 10grams. Other quantities for large scale production of stannsoporfin areat least about 25 grams, at least about 50 grams, at least about 100grams, at least about 200 grams, at least about 500 grams, at leastabout 1.0 kg, at least about 2.0 kg, or at least about 5.0 kg.

By “single batch” is meant that the amount of the product specified issynthesized at one time. A single batch is typically produced after areaction (or series of reactions) is carried out once (note that asingle preparation of the compound subjected as a whole to one or morereactions repeatedly, such as repeated purifications, is considered asingle batch). A single batch thus excludes multiple preparations of acompound carried out at separate times, or in divided amounts, which arelater combined.

By “high purity” is meant a preparation that meets both of the followingtwo criteria: 1) the overall level of purity is at least about 97%; thatis, the desired product (stannsoporfin) accounts for at least 97% of thepreparation; and 2) any individual product-related impurity present ispresent in an amount of less than about 0.1% of the preparation. Thepurity as preferably measured by HPLC analysis. A “product-related”impurity is an impurity that requires characterization by the guidelinesof the United States Food and Drug Administration; accordingly,components of the drug product such as water are not considered animpurity.

By “unmetallated porphyrin” is meant a porphyrin lacking a metal ioncoordinated by one or more pyrrole nitrogens. A “metallated porphyrin”is a porphyrin having a metal ion coordinated by at least one pyrrolenitrogen.

By “intermediate oxidation state” is meant an element, such as a metal,which is present in an oxidation state intermediate between its neutral(uncharged, or zero oxidation state) and its most highly oxidized state.By way of non-limiting example, iron typically forms oxidation states of(0), (II), and (III); the (II) oxidation state (ferrous state) is anintermediate oxidation state.

The purity of the preparation is important for use of the compound as apharmaceutical. The overall level of purity can be at least about 97%,at least about 93%, at least about 98.5%, at least about 99%, or atleast about 99.5%. A high-purity preparation is also defined, as above,as a preparation with the additional condition that any individualimpurity present is present in an amount of less than about 0.1% of thepreparation. (Note that the total amount of impurities may exceed0.1%—for example, one impurity may be present at 0.08%, and another at0.07%, totaling 0.15%—but when measured individually, no impurity ispresent at amounts equal to or exceeding about 0.1%.) In anotherembodiment, any individual impurity present is present in an amount ofless than about 0.09%. In another embodiment, any individual impuritypresent is present in an amount of less than about 0.08% or less. Inanother embodiment, any individual impurity present is present in anamount of about 0.07% or less. Water can be present in the preparation,even in significant amounts (at least about 1% to 5%), but it notconsidered an impurity. Other residual solvents, such as acetone, formicacid, and acetic acid, are also not considered impurities, especially ifthey occur at or below the permissible levels described in theguidelines of the International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for Human Use, ICHHarmonised Tripartite Guideline-Impurities: Guideline for ResidualSolvents, Q3C(R3), Step 4 version, November 2005 (World-WideWeb.ich.org/LOB/media/MEDIA423.pdf).

In an alternate embodiment, the stannsoporfin has no impurity present inan amount greater than about 0.2%, and more preferably has no impuritypresent in an amount greater than 0.15%, and still more preferably hasno impurity present in an amount greater than 0.12%.

The current synthesis produces stannsoporfin meeting the two criterialisted wider high-purity above (overall purity of at least about 97%,without treating water or residual solvents as impurities, and anyindividual impurity present is present in an amount of about 0.1% orless). The second criterion, regarding the level of individualimpurities, is of interest due to regulatory requirements. The Food andDrug Administration of the United States of America typically requiresdetailed characterization of impurities at a level equal to 0.1%, whileimpurities present at a level below 0.1% need not be characterized indetail unless they have unusually potent pharmacological or toxiceffects at a level of less than 0.1% (see the publications Guidance forIndustry: ANDAs: Impurities in Drug Substances, U.S. Department ofHealth and Human Services, Food and Drug Administration, Center for DrugEvaluation and Research (CDER), November 1999; available atWorld-Wide-Web-site-fda.gov/cder/guidance/2452fnl.htm; and Guidance forIndustry, Q3A Impurities in New Drug Substances, United StatesDepartment of Health and Human Services, Food and Drug Administration,Center for Drug Evaluation and Research (CDER) and Center for BiologicsEvaluation and Research (CBER), February 2003 ICH, Revision 1, availableat World-Wide-Web-site-fda.gov/cder/guidance4164fnl.pdf). By meetingthese threshold conditions set by the Food and Drug Administration, thehigh-purity material has significant advantages over material of lowerpurity from the regulatory standpoint.

Another advantage of the invention as described herein is the expectedreproducibility of the synthesis, providing the ability to generaterepeated batches of high-purity stannsoporfin in large quantity. Anotheradvantage of the invention as described herein is the ability of theprocess and product to meet requirements for Good Manufacturing Practice(GMP), as defined by law, regulation, or regulatory agency requirementsin various countries (for example, current Good Manufacturing Practiceas specified in the United States Code of Federal Regulations, Title 21,Sections 210 and 211).

Another advantage of the invention as described herein is the productionof high-purity bulk amounts of stannsoporfin in a single batch, withconcomitant advantages of increased homogeneity, lower synthetic cost,and relative ease of characterization.

Synthesis of Stannsoporfin at High Purity

As porphyrins are light-sensitive compounds, the starting materials,intermediates, products, and solutions or suspensions thereof should beprotected from light exposure, and stored in a dark location inlight-excluding containers.

Synthesis of stannsoporfin proceeds with hemin (iron (III)protoporphyrin IX chloride) as a starting material. The quantitiesrequired for large-scale synthesis are obtained from porcine red bloodcells. Hemin DMF grade is purchased from Harimex (Loenen, TheNetherlands); the material is used without purification prior to use(purity as supplied is greater than about 98% by HPLC). The hemin isheated in organic solvent with a hydrogenation catalyst on carbon undera hydrogen atmosphere. This reductive step serves both to remove the Feion from the porphyrin ring, and to reduce the protoporphyrin IX vinylgroups to ethyl groups (thus converting the protoporphyrin IX intomesoporphyrin IX), as indicated in the following scheme.

A preferred hydrogenation catalyst is palladium on carbon, used in anamount of about 0.0135 to 0.0165 equivalents, preferably about 0.015equivalents. Other suitable catalysts can be used, including palladiummetal particles, platinum on carbon, platinum metal particles, nickel,or nickel-aluminum catalyst, provided that residual amounts of catalystin the product meet pharmaceutical specifications. The nickel-aluminumcatalyst can be RANEY nickel (RANEY is a registered trademark of W.R.Grace & Co., New York, N.Y.). A preferred organic solvent is formicacid.

It was discovered that pre-treatment of the Pd/C catalyst with hydrogengas prior to addition of hemin to the reaction reduces palladiumimpurities and thus contributes to the overall purity of the finalstannsoporfin product. Without pre-hydrogenation of the catalyst priorto hemin addition, residual palladium levels of about 50 ppm weredetected in the product, which is significantly above the productspecifications of less than about 20 ppm residual palladium. With thepre-hydrogenation step, residual palladium was reduced to undetectablelevels (less than about 5 ppm residual palladium). Accordingly, theimproved synthesis provides for levels of residual palladium levels inthe product tin (IV) mesoporphyrin IX dichloride of less than about 20ppm palladium, preferably less than about 15 ppm palladium, morepreferably less than about 10 ppm palladium, still more preferably lessthan about 5 ppm palladium: The pre-hydrogenation of the catalyst can beperformed under a hydrogen atmosphere of about 15 to 75 psi (about 1 to5 bar; about 100,000 to 500,000 Pascals), preferably about 30 to 50 psi(about 2 to 3.5 bar; about 200,000 to 350,000 Pascals), more preferablyabout 40 psi (about 2.75 bar or 275,000 Pascals). The temperature forpre-hydrogenation of the catalyst can vary between about 25 to 60° C.,preferably about 35 to 50° C., more preferably about 40 to 45° C. Thetime period for pre-hydrogenation of the catalyst can range from about 2to 48 hours, preferably from about 6 to 24 hours, more preferably fromabout 8 to 16 hours, still more preferably about 12 hours.

Thus, typically, the catalyst is added to the chemical reactor first,followed by the formic acid solvent (for example, about 17.5 to 22.5parts solvent, preferably about 20 parts solvent). Before addition ofsolvent, the hydrogen can be evacuated and the reactor can be filledwith a nitrogen atmosphere for safety reasons. Upon completion of formicacid addition, the nitrogen atmosphere is replaced by a hydrogenatmosphere, at, for example, about 40 pounds per square inch(approximately 2.75 bar or 275,000 Pascals). The temperature is thenadjusted to about 35 to 50° C. preferably about 40 to 45° C., forapproximately 8 to 24 hours, preferably about 12 hours, prior tointroduction of the hemin starting material into the reactor. Thepre-hydrogenated catalyst suspension is then cooled, followed byaddition of hemin (in solvent) to the reactor. The hydrogen atmosphereis evacuated during the introduction of hemin for safety purposes,leaving only the hydrogen associated on the Pd/C catalyst. The reactoris re-pressurized to about 30 to 35psi with hydrogen, and the reactionis agitated at about 20 to 25° C. for about 30 minutes. The reaction isthen warmed to about 80 to 100° C., preferably to about 85 to 90° C.,with vigorous agitation, and hydrogen pressure is increased to about 50to 70 psi about 3.4 to 4.8 bar or about 340,000 to 480,000 Pascals),preferably about 55 to 60 psi (about 3.8 to 4.2 bar or about 380,000 to420,000 Pascals). The reaction temperature is maintained for about 1 to3 hours, preferably about 1 to 1.5 hours. The reaction is then cooled toabout 40 to 60° C., preferably to about 45 to 50° C., and hydrogenpressure maintained and hydrogenation continued for about 18 to 48hours, preferably 20 to 30 hours, more preferably about 24 hours.

The reaction is then cooled and depressurized with evacuation ofhydrogen from the reactor, Diatomaceous earth (such as HYFLO SUPERCEL, aregistered trademark of Celite Corp., Santa Barbara, Calif.), activatedcarbon (such as DARCO KB, a registered trademark of NORIT Americas,Inc., Marshall, Tex.), and solvent are added to the reactor. Thesuspension is filtered, and the filter cake is washed with solvent. Thistreatment serves to remove residual iron and residual palladium from thematerial.

The filtrate is concentrated by distillation under vacuum (which can beperformed at room temperature, or at lower temperatures, such as atapproximately 10 to 15° C.) to remove excess solvent. A precipitant, forexample, an ether such as methyl t-butyl ether (MTBE), is then added,over a period of at least about 30 seconds to at least about 3 hours,preferably over a period of at least about 1 hour, to the concentratedsolution. When MTBE is added, it can be added in about 17.5 to 22.5parts, preferably in about 20 parts.

The suspension can be cooled to a temperature of about −15 to −30° C.,preferably to about −20 to −25° C.

The suspension is filtered and the filtercake rinsed with an organicsolvent, such as ethers, including methyl t-butyl ether (MTBE), diethylether, or diisopropyl ether. After filtration is complete and the cakeis rinsed, the material is then dried in a vacuum oven at a temperaturenot exceeding about 60° C. for example, from about 45 to 60° C.

When prepared using formic acid as the solvent, the resulting product,mesoporphyrin IX, is precipitated out as a formate salt; this is thepreferred form for isolation of the mesoporphyrin IX after thehydrogenation step. After additional purification steps, themesoporphyrin IX formate is converted into a hydrochloride salt. Thisstep provides a further purification of the intermediate. In addition,the presence of proton scavengers such as formate for other organicanions, such as acetate) during, the subsequent tin insertion step hasbeen shown to result in higher levels of impurities than if suchscavengers are excluded. Accordingly, it is preferred to replace theformate anion of mesoporphyrin IX formate with an anion less capable ofscavenging protons or buffering the solution during the tin insertionstep; such anions include chloride and other halide anions such asbromide or iodide.

When the intermediate isolated from the hydrogenation step ismesoporphyrin IX formate, it is placed in a reaction vessel withdiatomaceous earth, activated carbon, and formic acid, (for example,with about 10% w/w diatomaceous earth, about 20% w/w activated carbon,and about 10 parts formic acid) to undergo additional purification. Thesuspension is agitated at, for example about 20 to 30° C. preferably atabout 20 to 25° C., for about 1.5 to 2.5 hours. The suspension is thenfiltered, and the filtercake washed with formic acid, for example, about5 parts of formic acid. The resulting filtrate solution is thenconcentrated down to about 5 to 6 parts volume. Another vessel ischarged with purified water and 31% hydrochloric acid to prepare about15 parts of approximately 1N hydrochloric acid. Approximately 6 parts ofthis HCl solution is transferred into the vessel containing about 6parts of the filtrate, preferably at a temperature of about 20 to 25° C.and over a period of at least about 60 minutes. The solution is thenseeded with mesoporphyrin IX dihydrochloride (available from earliersyntheses) and agitated, preferably for at least about 2 hours, Theremaining 9 parts of 1N hydrochloric acid is transferred into the vesselunder vigorous agitation, preferably over a period of at least 60minutes. The suspension is then further agitated at about 20 to 30° C.,preferably at about 20 to 25° C., for about 2 to 3 hours, it is thenfiltered, and rinsed with purified water. The product is dried on thefilter under a stream of nitrogen.

In earlier processes, the step above was carried out by re-dissolvingsolid mesoporphyrin IX formate in formic acid, and then adding theformic acid solution to the hydrochloric acid in order to convert themesoporphyrin IX formate to the mesoporphyrin IX dihydrochloride.However, filtration of the mesoporphyrin IX dihydrochloride so producedwas found to be quite slow on a pilot plant scale, requiring up to fivedays to complete, and the subsequent drying on the filter then tookbetween about two to three weeks. An improvement in the process wasdeveloped; as described above, the 1N hydrochloric acid solution isadded into the formic acid solution of mesoporphyrin IX formate. Thishas been discovered to result in mesoporphyrin IX dihydrochloride thatcan be filtered more rapidly. Addition of seed material can also beperformed during the procedure, for example, at the beginning of theaddition of the 1N HCl into the formic acid solution of mesoporphyrin IXformate, or during the addition of 1N HCl into the formic acid solutionof mesoporphyrin IX formate, such as when about 10%, about 20%, about30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%of the 1N HCl has been added to the formic acid solution ofmesoporphyrin IX formate. Preferably, as in the process as describedimmediately above, the seed material is added after 40% of the 1N HClhas been added to the formic acid solution of mesoporphyrin IX formate.The addition of the seed material can also aid in the formation of aproduct which can be filtered more quickly. Since the mesoporphyrin IXdihydrochloride resulting from these process improvements can befiltered much more quickly, on the order of hours or even minutesinstead of days, significant savings in time and cost are achieved.Thus, in another embodiment, the time for filtration of at least about10 grams of mesoporphyrin IX dihydrochloride is reduced to less thanabout 90 minutes, less than about 60 minutes, less than about 45minutes, less than about 35 minutes, less than about 25 minutes, or lessthan about 10 minutes. In a further embodiment, the time for filtrationof at least about 1000 grams of mesoporphyrin IX dihydrochloride isreduced to less than about 1 day less than about 12 hours, less thanabout 6 hours, less than about 4 hours, less than about 3 hours, or lessthan about 2 hours.

Conversion of Mesoporphyrin IX Hydrochloride to Stannsoporfin (Tin (IV)Mesoporphyrin IX) via Treatment with Tin (II) Salt

The mesoporphyrin IX hydrochloride is then treated with a tin (II) salt,such as SnCl₂ in an organic solvent, such as acetic acid, underoxidizing conditions, which yields the desired product, tin (IV)mesoporphyrin IX dichloride (stannsoporfin). For example, mesoporphyrinIX dihydrochloride and tin (II) chloride are placed in a vessel, andacetic acid is added at about 20 to 30° C., preferably at about 20 to25° C. The suspended reagents are agitated for at least about 30minutes. With vigorous agitation, the mixture is warmed under an inertatmosphere (such as nitrogen or argon) to reflux.

Once reflux has commenced, an atmosphere of approximately 6% oxygen innitrogen is introduced into the headspace of the vessel. The gas can befrom about 3% to about 22% oxygen; about 6% is preferred to minimizeexplosion hazards. The mixture is kept at reflux for about 100 to 130hours. The use of the 6% oxygen in nitrogen atmosphere in the headspaceinstead of sparging or bubbling the gas mixture through the liquid hasbeen found to be advantageous for increasing the yield of tin (IV)mesoporphyrin IX dichloride. Tin (II) can enter the porphyrin ring tocomplex with the nitrogens, and can also leave the porphyrin ring.However, tin (IV) which is not already bound to the nitrogens of theporphyrin ring cannot enter the ring to complex with the nitrogens. Inorder to generate tin (IV) mesoporphyrin IX, the tin (II) ion must enterthe porphyrin ring, and then undergo oxidation to tin (IV) in situ.Excessively rapid oxidation of the tin (II) ion will cause the insertionreaction to stall, which can lower yields significantly. Accordingly,proper control of the rate of oxidation is needed. Introducing oxygeninto the mixture via the interface between the solvent and theoxygen/nitrogen headspace atmosphere provides this control and leads toa reasonable rate of reaction with a good yield of final product.

The reaction mixture can optionally be sampled during the tin insertionstep by lowering the temperature to about 50 to 70° C., preferably about55 to 60° C., removing a sample, and returning the reaction to reflux.

After the tin insertion step, the reaction mixture is cooled, and (waterfor injection) grade water is added. The suspension is then filtered,and the filter cake washed with WFI water. The filter cake is thenplaced under vacuum for a minimum of 4 hours to remove residual water.

Conversion of Mesoporphyrin IX Dihydrochloride to Stannsoporfin (Tin(IV) Mesoporphyrin IX) via Treatment with Tin (II) Oxide

Tin can also be inserted into the mesoporphyrin IX ring via treatment ofmesoporphyrin IX dihydrochloride with tin (II) oxides. This reaction canproceed to completion in as short a time as two hours, compared to thefour days to three weeks required for tin insertion using the tin (II)salt method described above. A solution/suspension mesoporphyrin IXdihydrochloride in a suitable solvent, for example, formic acid oracetic acid, is added to a solution/suspension of tin (II) oxide in asuitable solvent, for example, acetic acid or formic acid. An exemplaryprocedure is described below, and also in the Examples.

The mesoporphyrin IX dihydrochloride is dissolved/suspended in formicacid at ambient temperature. As the solution or suspension will have adeep purple color, it is advantageous to pulverize the mesoporphyrin IXdihydrochloride into as fine a powder as possible to aid in dissolution.

The tin (II) oxide is suspended in acetic acid at ambient temperatureand stirred. After prolonged stirring, the tin oxide suspension maytransform into a gel, which was not observed to affect the reactionadversely. The gel breaks up once the addition of mesoporphyrin IXdihydrochloride commences. The amount of tin (II) oxide is about twoequivalents to about six equivalents per equivalent of mesoporphyrin IXdihydrochloride; preferably, about four equivalents of tin (II) oxideare used per equivalent of mesoporphyrin IX dihydrochloride. On thisreaction, the equivalent ratio is the same as the molar ratio.)

The tin (II) oxide solution is maintained at a temperature of about25-115° C., preferably about 50-75° C., more preferably about 60-65° C.The solution of mesoporphyrin IX dihydrochloride is then added over aperiod of about three to nine hours, preferably over a period of aboutsix hours. The solution of mesoporphyrin IX dihydrochloride can be atambient temperature during the addition, or can be maintained at atemperature of about 50-75° C. such as about 60-65° C. during addition.The reaction mixture is maintained at about 25-115° C., preferably about50-75° C., more preferably about 60-65° C., for about an additional 2 to48 hours, preferably about an additional 16 to 30 hours, more preferablyabout an additional 18 to 24 hours, such as about an additional 18 hoursor about an additional 24 hours. After the additional reaction time, thesuspension is cooled to room temperature (about 20-25° C.), agitated orstirred for at least about five minutes, preferably at least about onehour, and filtered.

General Metal Insertion into Porphyrins Using Metal Oxides

The procedure used for tin insertion into porphyrin rings using metaloxides can also be applied to insertion of other metals using metaloxides. Particularly useful metal oxides are metal oxides where themetal cation of the metal oxide is in an intermediate oxidation state.The procedure can be used for porphyrin compounds or salts thereof,including, but not limited to, a mesoporphyrin or a salt thereof,mesoporphyrin IX or a salt thereof, mesoporphyrin IX dihydrochloride, aprotoporphyrin or a salt thereof, a hematoporphyrin or a salt thereof,or a deuteroporphyrin or a salt thereof, to yield the metallatedporphyrin compound (or a salt thereof).

Metal oxides which can be used include, but are not limited to, tinoxide, zinc oxide, copper oxide, cadmium oxide, cobalt oxide, chromiumoxide, iron oxide, aluminum oxide, titanium oxide, nickel oxide,manganese oxide, silver oxide, gold oxide, vanadium oxide, platinumoxide, antimony oxide, arsenic oxide, tin (II) oxide, zinc (II) oxide,copper (I) oxide, copper (II) oxide, cadmium (II) oxide, cobalt (II)oxide, cobalt (III) oxide, cobalt (IV) oxide, Co₃O₄, chromium (II)oxide, chromium (III) oxide, chromium (IV) oxide, chromium (V) oxide,chromium (VI) oxide, iron (II) oxide, iron (III) oxide, Fe₃O₄, aluminum(III) oxide, titanium (II) oxide, titanium (III) oxide, titanium (IV)oxide, nickel (II) oxide, manganese (II) oxide, manganese (III) oxide,manganese (IV) oxide, manganese (VII) oxide, silver (I) oxide, silver(II) oxide, gold (I) oxide, gold (III) oxide, vanadium (II) oxide,vanadium (III) oxide, vanadium (IV) oxide, vanadium (V) oxide, platinum(II) oxide, platinum (IV) oxide, antimony (III) oxide, antimony (IV)oxide, antimony (V) oxide, arsenic (III) oxide, or arsenic (V) oxide.

Other porphyrin compounds and tetrapyrroles can also be metallated usingthe procedures described herein, including, but not limited to,porphyrins such as deuteroporphyrins and deutemporphyrin IX2,4-bis(ethylene glycol)(8,13-bis(1,2-dihydroxyethyl)3,7,12,17-tetramethyl-21H,23H-porphine-2,18-dipropionic acid). Additional porphyrin compoundswhich can be metallated using the procedures described herein include,but are not limited to, coproporphyrins, cytoprophyrins, etioprophyrins,hematoporphyrins, mesoprophyrins, phylloprophyrins, protoporphyrins,pyrroporphyrins, rhodoporphyrins, uroporphyrins, and phytoporphyrins. Acomprehensive listing of porphyrin compounds is given atWorld-Wide-Web.chem.qmul.ac.uk/iupac/tetrapyrrole/; the porphyrinsdescribed therein are hereby incorporated by reference herein asporphyrins which can be metallated using the procedures describedherein.

Purification of Tin (IV) Mesoporphyrin IX Dichloride: Hot AcidTrituration

At this point, the crude tin (IV) mesoporphyrin IX dichloride is thentriturated with hot acid in order to remove impurities. The material isre-suspended in hydrochloric acid (approximately 0.5N to 2.0N,preferably 1N) and the temperature raised to about 75 to 100° C. orabout 80 to 100° C., preferably about 85 to 95° C., more preferably toabout 85 to 90° C., for about one to two hours with moderate agitation.The suspension is then cooled to about 20 to 30° C., preferably to about20 to 25° C., and filtered; the filtercake is rinsed with purified waterand dried on the filter under a nitrogen stream.

Purification Tin (IV) Mesoporphyrin IX Dichloride: Treatment at High pH

The material from the hot acid trituration step is combined withdiatomaceous earth, activated carbon, water, and ammonium hydroxide. Thetemperature is adjusted to about 20 to 30° C., preferably to about 20 to25° C., and agitated, preferably for about 1 to 2 hours. A sample istaken to ensure that the pH is at or above approximately 9. The mixtureis then agitated, preferably for about 1 to 2 hours further. The mixtureis then filtered. Any material remaining on the filter is rinsed withwater; the filtercake is then discarded.

Re-Acidification of Tin (IV) Mesoporphyrin IX Dichloride

The filtrate is then transferred into a mixture of acetic acid and 31%hydrochloric acid, and the mixture is adjusted to about 20 to 30° C.,preferably to about 20 to 25° C. The resultant suspension is agitated,preferably for about 15 minutes, sampled to ensure that the pH is lessthan or equal to about 1, and then agitated again, preferably for aboutan additional 1 to 2 hours. The suspension is then filtered, and thefiltercake rinsed with water, followed by removal of residual waterunder vacuum.

At this stage, the filtercake is sampled for residual starting material,mesoporphyrin IX dihydrochloride. If the level is above about 0.1%, thehigh pH treatment followed by re-acidification is repeated as necessary(for example, an addition 1, 2, or 3 times).

Additional Hot Acid Trituration Tin (IV) Mesoporphyrin IX Dichloride

The filtercake from the previous step is re-suspended in a mixture ofapproximately two parts by weight WFI grade water and approximately onepart by weight 31% HCl, at about 20 to 30° C. preferably at about 20 to25° C. Under moderate agitation, the mixture is adjusted to about 80 to100° C., preferably to about 85 to 90° C. for about 6 to 48 hours,preferably about 12 to 24 hours, more preferably about 16 to 18 hours,followed by cooling to about 20 to 30° C., preferably to about 20 to 25°C., for at least about 1 hour. The suspension is filtered, thefiltercake rinsed with an aqueous solution of hydrochloric acid (forexample, about 1 part 31% HCl to 25 parts WFI grade water, w/w), anddried under a stream of nitrogen (at or below about 50° C.).

The final hot acid treatment serves in order to reset the form of thestannsoporfin to monomer. In neutral solution, stannsoporfin is in amonomer-dimer equilibrium; treatment with strong acid shifts theequilibrium strongly to the monomer form.

Development work on the stannsoporfin synthesis indicates that foroptimum results, the hydrogenation catalyst should be pre-hydrogenatedprior to introduction of the hemin starting material; the isolation ofthe mesoporphyrin IX dihydrochloride from the mesoporphyrin IX formatein formic acid should proceed by addition of the HCl solution to theformic acid solution; the presence of proton scavengers should beavoided during the tin insertion step; and the introduction of theoxygen during the tin insertion step should proceed via introduction ofthe oxygen/nitrogen mixture to the headspace of the reaction, ratherthan bubbling or sparging of the gas through the solution. With theseoptimum parameters in mind, other variables such as temperature,reaction time, reagent concentration, and order of reagent addition canbe manipulated to some extent, for example, concentration and reactiontime can be varied within about 50 to 200% of the values indicated, orwithin about 75 to 150% of the values indicated, and temperature can bevaried about 5 to 10° C. of the values indicated, to the extent that thevariation does not result in large-scale synthesis of stannsoporfin atless than high purity as defined herein. Purification and precipitationsteps can be repeated as necessary in order to maintain high purity ofthe large scale preparation of stannsoporfin.

Therapeutic Use of Stannsoporfin for Treatment or Prevention of InfantHyperbilirubinemia and Other Diseases

Stannsoporfin as produced by the invention can be used for treatment orprevention of infant hyperbilirubinemia (infant jaundice) (see U.S. Pat.Nos. 4,657,902; 4,668,670; and WO 94/28906). Additional methods of usingstannsoporfin are disclosed in U.S. Pat. No. 4,692,440 (to increase therate of heme excretion), WO 89/02269 (to counteract the toxicity ofcancer therapy), U.S. Pat. No. 4,782,049 (to treat psoriasis), and otherpublications. Treatment or prevention of infant hyperbilirubinemia isaccomplished by dissolving the stannsoporfin in a pharmaceuticallyacceptable vehicle. The stannsoporfin is preferably provided in asolution which can be buffered to maintain a suitable pH. Buffers whichcan be used include phosphate, citrate, gluconate, lactate, tartrate,glycinate, glcylglycinate, bicarbonate, carbonate, maleate, or acetate,with sodium, potassium, magnesium, calcium, or aluminum present as thecation, Histidine and imidazole can also be used as buffers. Phosphatebuffers are preferred, particularly sodium phosphate buffer. Buffersmust be pharmaceutically acceptable for use as an injectable agent inneonates. The pH of the solution for administration is preferablybetween about 7.0 to 8.0, more preferably about 7.2 to 7.9, still morepreferably about 7.4. The osmolarity of the solution is preferably at ornear physiological osmolarity; a preferred range is between about 280mOmsm/L and 310 mOsm/L. Stannsoporfin is preferably administered byinjection, more preferably by intramuscular injection. The stannsoporfinis administered in an amount sufficient to treat or prevent infanthyperbilirubinemia, typically about 4.5 mg/kg birthweight; U.S. patentapplication Ser. No. 11/867,581 filed on Oct. 4, 2007, and International(Patent Cooperation Treaty) Patent Application No. PCT/US07/021,486filed on Oct. 4, 2007, both of which claim priority to U.S. ProvisionalPatent Application No. 60/849,509, filed on Oct. 4, 2006, disclose amethod of treating infant hyperbilirubinemia using lower doses ofstannsoporfin, such as 1.5 mg/kg birthweight or 3.0 mg/kg birthweight.

U.S. Pat. No. 6,818,763, U.S. Patent Application Publication2004/0210048, and U.S. patent application Ser. No. 11/096,359 arespecifically hereby incorporated by reference herein in their entirety.

The following examples are intended to illustrate the invention, and arenot intended to limit the invention in any manner.

EXAMPLES Example 1 Exemplary Synthesis of High-Purity StannsoporfinInitial Conversion of Hemin to Mesoporphyrin IX

A 200 L glass lined vessel, pressure-rated to 150 psi, is charged with0.6 kg of 5% palladium on carbon and 71 kg of formic acid. With vigorousagitation, the reactor is pressurized with hydrogen to 60-65 psi andwarmed to 40-45° C. for a minimum of 12 hours. With moderate agitation,the reaction is cooled to 20-25° C., the hydrogen atmosphere isevacuated, and the reactor charged with 6.0 kg of hemin (DMF grade) and73 kg formic acid. The reactor is pressurized to 30-35 psi with hydrogenand agitated at 20-25° C. for 30 minutes.

With vigorous agitation the reaction is warmed to 85-90° C. Hydrogenpressure is then increased to 55-60 psi. The pressure and temperatureare maintained for a period of 1-1.5 hours.

The reaction is cooled to 45-50° C. and the hydrogenation is continuedat 55-60 psi for 24 hours. The reaction is then cooled to 20-25° C.,depressurized and sampled.

The reaction is warmed to 45-50° C., pressurized to 55-60 psi withhydrogen, and agitated a further 6 hours. The reaction is then cooled to20-25° C., depressurized and sampled again.

Hydrogen is evacuated from the vessel, which is then charged with 3.0 kgHYFLO SUPERCEL, 2.3 kg DARCO KB and 42 kg formic acid. The suspension isfiltered, and the filter cake is rinsed with 122 kg formic acid.

A portion of the filtrate is transferred to a 200 L glass lined vessel,cooled to 10-15° C. and distilled under vacuum to remove formic acid.Once the residual volume has dropped to 25-35 L, the remainder of thefiltrate is transferred in and distillation continued to a residualvolume of 25-30 L.

The reaction to temperature is adjusted to 20-25° C. and 89 kg of methyltert-butyl ether is added over a minimum of 1 hour. The resultantsuspension is agitated at 20-25° C. for 2 hours prior to cooling to −25to −20° C. for a period of 4 hours.

The suspension is filtered and rinsed with 12 kg of methyl tert-butylether. The intermediate product is dried in a vacuum oven at 60° C. orless.

Purification of Mesoporphyrin IX Formate with Diatomaceous Earth andActivated Carbon; Conversion of Mesoporphyrin IX Formate toMesoporphyrin IX Dihydrochloride

The intermediate is transferred to a 50 L glass lined vessel with 10%w/w DARCO KB, 20% w/w HYFLO SUPERCEL and 10 parts formic acid. Thesuspension is agitated at 20-25° C. for a period of 1.5-2.5 hours.

The suspension is filtered into a second 50 L glass lined vessel. Thefiltercake is rinsed with 5 parts formic acid and discarded. Thefiltrate solution is vacuum distilled to a residual volume of 5-6 parts.

A third vessel is charged with purified water and 31% hydrochloric acidto prepare 15 parts of 1N hydrochloric acid. Six parts of the filtratesolution is transferred into the reactor at 20-25° C. over a minimum of60 minutes.

The solution is seeded with mesoporphyrin IX dihydrochloride andagitated for a minimum of 2 hours. With vigorous agitation, theremaining 9 parts of 1N hydrochloric acid is transferred into the vesselover a minimum of 1 hour.

The resultant suspension is agitated at 20-25° C. for a period of 2-3hours prior to isolation by filtration. The filtercake is rinsed with 4parts of purified water. The intermediate product mesoporphyrin IXdihydrochloride is dried on the filter under a stream of nitrogen.

Conversion of Mesoporphyrin IX Dihydrochloride to Tin (IV)Mesomporphyrin IX Dichloride (Stannsoporfin)

A 50 L glass lined vessel is charged with 1.57 kg of mesoporphyrin IXdihydrochloride, 1.862 kg of tin (II) chloride, and 40.9 kg acetic acidat 20-25° C. With moderate agitation, the suspension is maintained at20-25° C. for a minimum of 30 minutes.

With vigorous agitation, under nitrogen, the mixture is warmed to reflux(ca. 115° C.). Once reflux has been attained, a 6% oxygen in nitrogenatmosphere is introduced to the headspace of the vessel. The reactionmixture is maintained at reflux for a period of 100-130 hours.

The reaction mixture is cooled to 55-60° C. and sampled for residualmesoporphyrin; while awaiting results, the reaction mixture is warmedback to reflux. Once complete, the reaction is cooled to 60-70° C. andcharged with 15.7 kg of WFI (water for injection) grade water. Thetemperature of the suspension is adjusted to 20-25° C. over 30 minutesand agitated for a period of 1 hour.

The suspension is filtered, and the vessel and cake are rinsed with 6.3kg of WFI water. Upon completion of the wash, the cake is placed undervacuum for a minimum of 4 hours to remove residual water.

A 50 L glass lined vessel is charged with the wet filtercake 22.4 kgpurified water, and 3.7 kg 31% hydrochloric acid at 20-25° C. Withmoderate agitation, the temperature of the mixture is adjusted to 85-90°C for a period of 1-2 hours, followed by cooling to 20-25° C. Thesuspension was filtered and the filtercake rinsed with 6.3 kg purifiedwater. The product is dried on the filter under a stream of nitrogen andpackaged.

Purification of Tin (IV) Mesoporphyrin IX Dichloride (Stannsoporfin) atHigh pH with Diatomaceous Earth and Activated Carbon

A 50 L glass lined vessel is charged with 1.448 kg of tin (IV)mesoporphyrin IX dichloride, 0.194 kg HYFLO SUPERCEL, 0.066 kg DARCO KB,14.5 kg WFI water, and 1.0 kg ammonium hydroxide 26 Be. The temperatureof the reaction mixture is adjusted to 20-25° C. and agitated for aperiod of 1-2 hours. A sample is taken to verify that the pH is ≥9. Themixture is then agitated a further 1-2 hours. The mixture is filteredthrough into a glass receiver. The cake rinsed with 2.9 kg of water anddiscarded.

A second 50 L glass lined vessel is charged with 38.2 kg of acetic acidand 2.5 kg of 31% HCL. The temperature is adjusted to 20-25° C. Thefiltrate from the glass receiver is transferred into the second 50 Lvessel over a minimum of 45 minutes at 20-25° C. The glass receiver andtransfer apparatus are rinsed with 2.1 kg WFI water into the vessel. Theresultant suspension is agitated at 20-25° C. for 15 minutes prior totaking a sample to verify that the pH is ≤1. The suspension is thenagitated a further 1-2 hours.

The suspension is filtered, and the vessel and cake are rinsed with 1.3kg of WFI water. Upon completion of the wash, the cake is placed undervacuum for a minimum of 4 hours to remove residual water.

A sample of the filtercake is taken for testing. If the residualstarting material (mesoporphyrin IX dihydrochloride) is at an acceptablelevel, the reaction proceeds to the next step, otherwise the entiretreatment is repeated (i.e., the filtercake is re-dissolved usingammonium hydroxide as above.

Treatment of Tin (IV) Mesoporphyrin IX Dichloride (Stannsoporfin) at LowpH to Set Monomer Form

The wet filtercake is returned to the 50 L glass lined vessel which isthen charged with 2.4 kg WFI water and 10.2 kg 31% hydrochloric acid at20-25° C. With moderate agitation, the temperature of the mixture isadjusted to 85-90° C. for a period of 16-18 hours, followed by coolingto 20-25° C. for a minimum of 1 hour. The suspension is filtered and thefiltercake rinsed with a pre-mixed solution of 0.5 kg 31% hydrochloricacid in 12.8 kg WFI water. The product is dried on the filter at <50° C.under a stream of nitrogen and packaged.

Example 2 Alternative Tin Insertion Step Using Tin (II) Oxide as TinSource

The insertion of tin into mesoporphyrin IX to produce stannsoporfin canbe carried out by an alternate synthetic route using tin (II) oxide asthe reagent for tin introduction.

A dark, 1000 ml, three-necked, round-bottom flask equipped with amagnetic stirbar, Claisen head, addition funnel, thermometer, condenser,and nitrogen bubbler was charged with 8.4 g tin (II) oxide, and 200 mlacetic acid, at 20-25° C., to form a gray suspension. The suspension waswarmed to 60-65° C. under nitrogen.

A separate 250 ml one-neck, round-bottom flask equipped with a stirbarwas charged with 10 g mesoporphyrin IX dihydrochloride, and 50 ml formicacid. The mixture was agitated at 20-25° C. for 30 minutes to effectdissolution, resulting in about 60 ml of a deep purple suspensionsolution at 20-25° C. (Because of the colored solution, completedissolution is difficult to observe visually; the mesoporphyrin IXdihydrochloride should be thoroughly milled prior to formic acidaddition.)

The mesoporphyrin IX dihydrochloride solution was charged to theaddition funnel and added dropwise to the tin (II) oxide/acetic acidsuspension solution over a period of 6 hours, while maintaining thetemperature of the tin (II) oxide/acetic acid suspension/solution at60-65° C. The volume in the flask increased from 200 ml to 260 ml; theappearance of the reaction changed from a gray suspension (or whitegel), to a purple suspension, to a red suspension.

Once the addition was complete, the reaction was agitated under nitrogenatmosphere at 60-65° C. for a further 18-24 hours. Then 100 ml water wasadded dropwise over 20-40 minutes, while maintaining, the temperature at60-65° C. The resultant red suspension (about 360 ml) was cooled to20-25° C. over 30 minutes and agitated for a minimum of 1 hour, followedby filtration under reduced pressure (total filtration time was about10-20 minutes). The filtercake was rinsed with two portions of 20 mlwater. The filtrate volume of about 400 ml was a claret-coloredsolution; the filtercake mass of about 40-50 g was also claret-colored.

The wet filtercake was carefully broken up into pieces and charged backinto the reaction flask with 100 ml 1N HCl. The resultant claret-coloredsuspension was warmed to 85-95° C. for 1 hour. The suspension was thencooled to 20-25° C. and filtered under reduced pressure (totalfiltration time was about 20-30 minutes); the filtrate was dark claretto brown in color. The claret-colored filtercake was rinsed with twoportions of 20 ml water, dried under a nitrogen stream, and furtherdried under high vacuum at 80-90° C. for 24 hours. In variousrepetitions of the synthesis, the yield of product varied from 16.5-21.2g (70-90%).

Example 3 Analysis of High-Purity Stannsoporfin Made by ExemplarySynthesis Utilizing Tin (II) Chloride as Tin Source

Batches of stannsoporfin were prepared using the exemplary synthesisessentially as outlined above in Example 1, as well as earlier methods(see U.S. Pat. No. 6,818,763 and U.S. 2004/0210048).

Basic HPLC analysis is performed using a C-18 column (Zorbax ExtendC-18, 4.6×150 mm, 3.5 μm particle size, or the equivalent). The detectoris set at 400 nm. Solvents (acetonitrile, methanol, and water) are HPLCgrade. The mobile phase is 16% acetonitrile: 40% methanol: 44% 0.5 Mammonium acetate, pH 5.15. (The ammonium acetate solution is prepared bydissolving 38.5 g ammonium acetate in 440 mL H₂O, and adjusting the pHto 5.15 with acetic acid. Both the ammonium acetate and acetic acid arereagent grade. 160 mL acetonitrile and 400 mL methanol are then added;the mobile phase solution is mixed, filtered, and degassed prior touse.) The flow rate is 1.0 ml/minute. Samples and standards ofstannsoporfin are prepared for injection at a concentration of 0.04mg/mL in 1N NaOH. As stannsoporfin and related compounds are lightsensitive, solutions containing stannsoporfin, starting materials, orimpurity standards should be kept in opaque containers, and handling andanalysis should be conducted under reduced light conditions. Samples andstandard solutions should be used within 12 hours of preparation. 5 μLof analyte solution is injected, and a 10-minute run time is used. Theretention time of stannsoporfin is typically about 4.8 minutes. Thecolumn temperature is maintained at 60° C. After analysis, the column iswashed with 80% methanol and 20% water for at least 1 hour at 1.0mL/min.

HPLC analysis for quantitation of impurities is performed using an ACE 5C-18 column, 4.6×250 mm. 5 μm particle size, with detection at 400 nm.Protection of light-sensitive samples and standards is practiced asdescribed above. The mobile phases used are A: 30% methanol, 70% waterwith 0.02 M ammonium acetate, pH 9.1, and B: 80% methanol, 20% waterwith 0.02 M ammonium acetate, pH 9.1 (mobile phase A is prepared bydissolving 3.0 g of ammonium acetate in 1400 mL water, adjusting pH to9.1 with NH₄OH, and adding 600 mL methanol; mobile phase B is preparedby dissolving 3.0 g of ammonium acetate in 400 mL water, adjusting pH to9.1 with NH₄OH, and adding 1600 mL methanol; mobile phases are mixed,filtered, and degassed prior to use). Samples are dissolved in 0.5% v/vTEA in water at a concentration of approximately 0.2 mg/mL. Samples andstandard solutions should be used within 12 hours of preparation.

The analysis is performed using the following gradient conditions:

Time % A % B 0 100 0 50 70 30 65 70 30 90 0 100 110 0 100 111 100 0 120100 0where the concentrations are changed linearly between the points shown.

Table 1 contains a comparison of the HPLC analysis of the product of thecurrent synthesis, column C, as compared to analyses of products fromearlier syntheses in column A and column B. Peaks detected are listed inorder of retention time relative to stannsoporfin, with the retentiontime of stannsoporfin set to 1. The batch analyzed in column A wasproduced in a quantity of 1.1 kg; the batch analyzed in column C wasalso produced in a quantity of 1.1 kg.

TABLE 1 Analysis of various preparations of stannsoporfin RelativeRetention Time A B C 0.33 0.06% 0.51 0.05% 0.05% 0.07% 0.55 0.06% 0.730.14% 0.05% 0.06% 0.76 0.07% 0.05% 0.83 0.05% 0.84 0.05% 0.92 0.26%0.06% 0.95 0.30% 0.05% 0.96 0.22% 1   98%   99%  100% 1.05 0.09% 1.260.06%As seen from Table 1, the current stannsoporfin synthesis in column Cresulted in material which resulted in a high purity product, of overallpurity >99% and which does not contain any impurities at or above 0.1%.

Example 4 Analysis of High-Purity Stannsoporfin Made by ExemplarySynthesis Utilizing Tin (II) Oxide as Tin Source

Three batches of stannsoporfin were made using the tin insertion step asdescribed in Example 2. Analysis of the three batches indicated that thepurity of stannsoporfin produced was 99.7%, 99.7%, and 99.6% (totalcontent of stannsoporfin was 96.4%, 99.1%, and 97.2%, respectively).

HPLC analysis was performed on a Zorbax Extend C-18 column, 4.6×150 mm,5 μm thickness. The eluents used were: A: 80% 0.05 M Ammonium Acetate,pH 5.15 with Acetic Acid: 20% Acetonitrile; B: 90% Methanol: 10%Acetortitrile. The temperature used was 40° C. A flow rate of 1.2 ml/minwas used, with detection at 400 nm. The retention time of stannsoporfinwas 8.8 min, while that of mesoporphyrin IX was 23.1 min, with use ofthe following gradient listed in Table 2.

TABLE 2 Time A B 0.0 60 40 10.0 25 75 30.0 25 75 31.0 60 40 40.0 60 40

More extensive analyses of two batches of stannsoporfin produced usingthe tin oxide insertion method were conducted. These analyses aredetailed in Table 3 (batch weight 0.840 kg) and Table 4 (batch weight1.364 kg) below (where a/a indicates ratio of area of HPLC peaks).

TABLE 3 Test Method Results Total Purity HPLC Total impurities < 1% a/a;impurity at RRt 0.72 = 0.06% a/a; no other impurity > 0.05% a/a WaterContent Karl Fischer, coulometric Trace < 1% w/w Residual Solvents -Chromatographic (GC - Not detected < 0.1% w/w Acetone headspace) Organiccontent - HPLC 0.1% w/w formic acid + acetic acid Inorganic content -Inductively coupled Pd = 5 ppm palladium and iron plasma - opticalemission Fe = 5 ppm spectroscopy Inorganic content - Differential Pulse<0.1% free tin free tin Polarography Inorganic content - Inductivelycoupled 144500 ppm tin plasma - optical emission spectroscopy Inorganiccontent - Elemental analysis 104100 ppm chloride

TABLE 4 Test Method Results Total Purity HPLC Total impurities < 1% a/a;impurity at RRt 0.72 = 0.06% a/a; no other impurity > 0.05% a/a WaterContent Karl Fischer, coulometric Trace < 1% w/w Residual Solvents -Chromatographic (GC - Not detected < 0.1% w/w Acetone headspace) Organiccontent - HPLC Not detected < 0.1% w/w formic acid + acetic acidInorganic content - Inductively coupled Pd = 5 ppm palladium and ironplasma - optical emission Fe = 67 ppm spectroscopy Inorganic content -Differential Pulse <0.1% free tin free tin Polarography Inorganiccontent - Inductively coupled 165000 ppm tin plasma - optical emissionspectroscopy Inorganic content - Elemental analysis 103300 ppm chloride

Example 5 Stability of High-Purity Stannsoporfin Preparations

The long-term stability of the compound was studied under two differentstorage conditions: 25° C. (+/−2° C. and 60% relative humidity (+/−5%);and 40° C. (+/−2° C.) and 75% relative humidity (+/−5%). Primarypackaging for the compound was a 4-mil polyethylene bag and secondarypackaging for the compound was a 4 mil polyethylene bag, stored in anHDPE drum.

Table 5 and Table 6 show stability data for the batch described in Table3, under the 25° C./60% RH and 40° C./75% RH conditions, respectively,Table 7 and Table 8 show stability data for the batch described in Table4 under the 25° C./60% RH and 40° C./75% RH conditions, respectively.Data for the zero-month time point was taken from the batch releaseanalysis (the zero time point represents the actual date when sampleswere placed in the stability test chambers). The samples were analyzedat approximately 3 months and approximately 6 months after the sampleswere placed under the storage conditions.

TABLE 5 Test 0 months 3 months 6 months Appearance Red Red Red powderfree powder free powder free from visual from visual from visualevidence of evidence of evidence of contamination contaminationcontamination HPLC purity (total 0.3 0.22 0.24 impurities) HPLC purity(impurity  0.06% <0.05%  0.07% peak at RRt retention time 0.72-0.73)HPLC assay (w/w, 100.7% 99.8% 98.4% solvent-free anhydrous basis) HPLCassay (w/w as 100.4% 99.6% 98.2% is) Water content (Karl Trace < 1% <1%(0.1%) <1% (0.1%) Fischer, coulometric)

TABLE 6 Test 0 months 3 months 6 months Appearance Red Red Red powderfree powder free powder free from visual from visual from visualevidence of evidence of evidence of contamination contaminationcontamination HPLC purity (total 0.3 0.28 0.26 impurities) HPLC purity(impurity  0.06% <0.05% 0.06% peak at RRt retention time 0.72-0.73) HPLCassay (w/w, 100.7% 101.2% 99.8% solvent-free anhydrous basis) HPLC assay(w/w as 100.4% 100.9% 99.6% is) Water content (Karl Trace < 1% <1%(0.2%) <1% (0.1%) Fischer, coulometric)

TABLE 7 Test 0 months 3 months 6 months Appearance Red Red Red powderfree powder free powder free from visual from visual from visualevidence of evidence of evidence of contamination contaminationcontamination HPLC purity (total 0.22 0.29 0.19 impurities) HPLC purity(impurity  0.06% 0.05% 0.05% peak at RRt retention time 0.72-0.73) HPLCassay (w/w, 102.3% 102.1% 98.5% solvent-free anhydrous basis) HPLC assay(w/w as 102.3% 102.0% 98.4% is) Water content (Karl Trace < 1% <1%(0.1%) <1% (0.1%) Fischer, coulometric)

TABLE 8 Test 0 months 3 months 6 months Appearance Red Red Red powderfree powder free powder free from visual from visual from visualevidence of evidence of evidence of contamination contaminationcontamination HPLC purity (total 0.22 0.29 0.24 impurities) HPLC purity(impurity  0.06% <0.05% 0.06% peak at RRt retention time 0.72-0.73) HPLCassay (w/w, 102.3% 101.1% 97.8% solvent-free anhydrous basis) HPLC assay(w/w as 102.3% 101.0% 97.7% is) Water content (Karl Trace < 1% <1%(0.1%) <1% (0.1%) Fischer, coulometric)

The disclosures of all publications, patents, patent applications andpublished patent applications referred to herein by an identifyingcitation are hereby incorporated herein by reference in their entirety.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is apparent to those skilled in the art that certainminor changes and modifications will be practiced. Therefore, thedescription and examples should not be construed as limiting the scopeof the invention.

1-32. (canceled)
 33. A composition comprising stannsoporfin, wherein thestannsoporfin is at least about 97% pure, and wherein any individualimpurity is present in an amount less than about 0.08%.
 34. Thecomposition of claim 33, wherein the stannsoporfin is at least about98.5% pure.
 35. The composition of claim 33, wherein the compositioncomprises palladium in an amount of less than about 20 ppm.
 36. Thecomposition of claim 33, wherein the composition comprises palladium inan amount of less than about 10 ppm.
 37. The composition of claim 33,wherein any impurity present is present in an amount of about 0.07% orless.
 38. The composition of claim 33, wherein the composition has a pHof about 7.0 to about 8.0.
 39. The composition of claim 33, wherein thecomposition has a pH of about 7.2 to about 7.9.
 40. The composition ofclaim 33, comprising at least about 10 grams of stannsoporfin.
 41. Thecomposition of claim 33, comprising at least about 100 grams ofstannsoporfin.
 42. The composition of claim 33, comprising at leastabout 10 grams of stannsoporfin, wherein the composition containspalladium in an amount of less than about 20 ppm.
 43. The composition ofclaim 42, wherein the stannsoporfin is a single-batch preparation ofstannsoporfin.
 44. The composition of claim 33, wherein the compositionfurther comprises at least one impurity in an amount between about 0.05%and about 0.08%, the at least one impurity having a relative retentiontime of 0.72-0.73 in an HPLC analysis.
 45. A pharmaceutical compositioncomprising stannsoporfin and a pharmaceutically acceptable carrier,wherein the stannsoporfin is at least about 97% pure, and wherein anyindividual impurity is present in an amount less than about 0.08%. 46.The pharmaceutical composition of claim 45, wherein the stannsoporfin ispresent in an amount suitable for delivering up to a 4.5 mg/kg dose bybirth weight.
 47. The pharmaceutical composition of claim 45, whereinthe stannsoporfin is present in an amount suitable for delivering a doseof about 1.5 mg/kg to about 3.0 mg/kg by birth weight.
 48. Thepharmaceutical composition of claim 45, wherein the pharmaceuticalcomposition has a pH of about 7.0 to about 8.0.
 49. The pharmaceuticalcomposition of claim 45, wherein the pharmaceutical composition has a pHof about 7.2 to about 7.9.
 50. The pharmaceutical composition of claim45, wherein the stannsoporfin is at least about 98.5% pure.
 51. Thepharmaceutical composition of claim 45, wherein the pharmaceuticalcomposition comprises palladium in an amount of less than about 20 ppm.52. The pharmaceutical composition of claim 45, wherein thepharmaceutical composition comprises palladium in an amount of less thanabout 10 ppm.